YH206 cell line and monoclonal antibody produced by it

ABSTRACT

A monoclonal antibody specifically reactive to a human adenocarcinoma cell, which is obtainable by cultivating a fused cell obtained by fusing a mammalian animal cell, immunized with a human adenocarcinoma cell, with a myeloma cell.

This application is a continuation of application Ser. No. 06/798,276,filed Nov. 12, 1985, abandoned.

The present invention relates to a monoclonal antibody, and itspreparation and use. More particularly, it relates to a novel monoclonalantibody specifically reactive to a specific antigen in humanadenocarcinoma cells or leaked out therefrom into blood, a process forpreparing said monoclonal antibody by cultivation of hybridoma cells ofmyeloma cells and mammalian animal cells immunized with humanadenocarcinoma cells, and a method for immuno-chemical determination ofsaid specific antigen in sera by the use of said monoclonal antibody.

In recent years, there has been developed a process for preparation of amonoclonal antibodies in a large quantities which comprises cultivatinghybridoma cells obtained by cell fusion of antibody-producing cells withmyeloma cells (Milstein et al.: Nature, 256, 495-497 (1975)). Themonoclonal antibody to a cancer-related antigen obtained by the processas disclosed in this literature can be used not only for analysis of thecancer-related antigen itself but also for detection of the antigen inserum, radioimmuno-imaging diagnosis, etc. due to its high specificityand uniformity.

Hitherto, there have been reported some processes for preparation of amonoclonal antibody to human lung carcinoma. However, the reportsrelating to an antibody having a specificity to adenocarcinoma are few,and the successful detection of any antigen in blood has never beenreported. Recently, Iguro et al. reported that the monoclonal antibodyCSLEXI prepared for stomach cancer cells is effective in detection of anantigen in sera of patients bearing cancers such as lung carcinoma(Lancet, 6, 817-818 (1984)), but the molecule of such antigen has notsufficiently been analyzed. Further, the antibody has a cross-reactivitywith normal cells such as multinuclear leukocytes, liver cells andKupffer cells other than cancer cells so that it is hardly usable fordiagnosis of human cancers.

The monoclonal antibody 19-9 as ascertained by Koprowski et al. canrecognize a blood group-related antigen (Somatic Cell Genetics, 3,952-972 (1979)) and is used clinically for detection of a novel tumormarker. However, this antibody is positive not only to pancreas cancerand stomach cancer but also to colon cancer (59%). Among lung cancers,it is reactive to squamous epithelium carcinoma (3 cases in 4 cases) andsmall cell carcinoma (4 cases in 9 cases) in addition to adenocarcinoma.Thus, it is not specific to adenocarcinoma.

As a result of extensive study, there has now been successfully obtaineda monoclonal antibody which is specifically reactive to adenocarcinomasuch as lung adenocarcinoma, stomach adenocarcinoma and pancreasadenocarcinoma and hardly reactive to non-adenocarcinoma such assquamous epithelium carcinoma, large cell carcinoma and small cellcarcinoma as well as normal cells. It has also been found that thediagnosis of various adenocarcinoma can be successfully carried out byimmuno-chemical measurement of the specific antigen leaked out into theblood with said antibody.

According to the present invention, there is provided a monoclonalantibody having a specific reactivity to human adenocarcinoma cells,said antibody being produced from hybridoma cells obtainable by cellfusion of myeloma cells and mammalian animal cells immunized with humanadenocarcinoma cells. Quite surprisingly, the specific antigencorresponding to said antibody leaks out into the blood from variousadenocarcinoma cells. It is therefore possible to make the diagnosis ofvarious cancers by immuno-chemical measurement of the specific antigenin the blood with said antibody. Further, said antibody ischaracteristic in reacting specifically with human adenocarcinoma cellsand can, for instance, react with human lung cancer cells, especiallylung adenocarcinoma. The antibody is thus useful for distinguishingvarious lung cancers. Furthermore, the blood level of the antigencorresponding to said antibody increases with the progress of thesymptomatic stage of lung cancer so that the antibody can be used forinvestigation of the progressive degree of the cancer.

As stated above, the monoclonal antibody of the invention is not onlyuseful as a reagent for the biochemical and pathological study ofvarious cancers but also useful as a diagnostic agent for variouscancers by clinical measurement of specific antigen in the sera ofpatients. Further, said antibody may be labeled with a radioactiveisotope such as technetium-99 m or indium-111. Its administration topatients, followed by application of immuno-detection thereto accordingto the nuclear medical procedure makes the morphological diagnosticimaging of various cancers possible. Furthermore, said antibody may beconjugated with a radioactive isotope (e.g. iodine-131, yttrium-90) or acytotoxic agent. Its administration to patients would produce atherapeutic effect.

Preparation of the monoclonal antibody according to the presentinvention will be hereinafter explained in detail.

For attaining this object, it is necessary first to produce hybridomacells by cell fusion of antibody-producing cells with myeloma cells, andthis can be done by a per se conventional procedure such as the Milsteinet al. method as hereinabove stated.

The antibody-producing cells can be obtained by immunizing mammaliananimal cells with human lung adenocarcinoma cells as the antigen. Thehuman lung adenocarcinoma cells are not limitative, and any culturedhuman lung adenocarcinoma cells as already ascertained may be used.Especially preferred are human lung adenocarcinoma A549 cells. Themammalian animal cells are also not limitative, and their selection maybe made appropriately taking into consideration their adaptability withmyeloma cells used for cell fusion. In general, murine cells are usedfor this purpose. Immunization may be carried out by a per seconventional procedure. For instance, human cancer cells are dilutedwith a physiological saline solution and optionally admixed withFreund's complete adjuvant to make a suspension, which is administeredintracutaneously into animals. Administration is effected several timesat appropriate intervals. On day 3 or 4 after the last administration,animals having a high antibody titer are chosen, and the spleen cellsare taken out for the use as the immuno-competent cells, i.e.antibody-forming or antibody-producing cells.

The thus produced antibody-producing cells and the myeloma cells aresubjected to cell fusion. As the myeloma cells, there may be employedvarious ones chosen from P3/X63-Ag8, X63-Ag8.653, P3/X63-Ag-8.U1,P3/NS1-1-Ag4-1, SP2/0-Ag14, FO, MPC11-45.6TG1.7, etc. The cell fusionmay be accomplished by a per se conventional procedure, for instance, bymixing together the antibody-producing cells and the myeloma cells ascultured in appropriate culture media, followed by stirring, shakingand/or centrifuging. If necessary, feeder cells chosen from thymuscells, abdominal cavity exuding cells, spleen cells and the like, fusionpromoting agents such as polyethylene glycol, etc. may be added thereto.The culture medium may be any one as conventionally employed for cellculture, and its examples are an MEM culture medium, an RPMI-1640culture medium, etc.

The thus obtained hybridoma cells are cultivated in a conventionalmedium for selection of hydridoma such as an HAT medium so as to obtainthe desired hybridoma cells. Since the myeloma cells are deficient inhypoxanthin-guanine-phosphoribosyl transferase, they can not grow in theHAT medium. Further, the cells other than the desired hybridoma cellsbetween the antibody-producing cells and the myeloma cells, such as thenon-fused cells and the hybridoma cells of the antibody-producing cellscan live in the HAT medium only for about 2 weeks. Accordingly, only thedesired hybridoma cells can be recovered from the HAT medium aftercultivation therein over a certain period of time. The recoveredhybridoma cells are then subjected to collection of theantibody-producing strain and cloning by a conventional limitingdilution method.

The resultant monoclonal antibody-producing hybridoma cells can becultivated successively in a conventional culture medium. Also, they canbe stored in liquid nitrogen over a long period of time. The hybridomacells producing the monoclonal antibody YH206 (hereinafter referred toas "hybridoma YH206") are stored and retained by the inventorsthemselves and can be freely obtained from them.

Examination as to whether the hybridoma YH206 can produce the monoclonalantibody (hereinafter referred to as "monoclonal antibody YH206") may beeffected by any per se conventional antibody-detecting procedure such asthe indirect immuno-fluorescent method, the indirect immuno-peroxidasemethod, the enzyme antibody method (enzyme-linked immunosorbent assay(ELISA)) or the radioimmunoassay method.

For production of the monoclonal antibody YH206 with the hybridoma YH206cells, those hybridoma cells may be subjected to tissue culture,followed by collection of the culture supernatant. Alternatively, thehybridoma cells may be administered intraperitoneally to animals havingthe adaptability thereto for growth, and then the ascitic fluid may becollected therefrom. In the former process, the antibody having a highpurity is obtainable. In the latter process, the antibody can beproduced on a large scale. The collected supernatant or ascitic fluidmay be purified by a method as conventionally employed for purificationof proteins to give the desired monoclonal antibody YH206.

The immuno-histological diagnosis with the monoclonal antibody YH206 andthe diagnosis of cancer by measurement of the corresponding specificantigen (tumor marker) in the body fluid will be hereinafter explained.

Various cancer tissues are fixed with formalin and embedded in paraffin,followed by slicing. With the resulting specimens, the immuno-peroxidasemethod such as the anti-peroxidase complex method or theavidin-biotin-peroxidase complex method is effected, whereby thehistological diagnosis of cancer can be readily accomplished within ashort period of time without the complex procedure required forconventional pathologic histological observation.

The diagnosis of various cancers can be also accomplished by utilizationof the specific antigen corresponding to the monoclonal antibody YH206,said antigen being leaked out from the cancer cells into the body fluid.For instance, the quantity of the specific antigen exuded from thecancer cells into the body fluid may be determined by immunoassay withthe monoclonal antibody YH206, and the diagnosis of cancer is made fromthe obtained results. As the immunoassay, there may be adopted thereversed passive hemagglutination method, the enzyme immunoassay method,the fluoroimmunoassay method, the radioimmunoassay method or the like.

Practical and presently preferred embodiments of the invention areillustratively shown in the following Examples.

EXAMPLE 1

Preparation and screening of the hybridoma YH206:

Into each of BALB/c mice, human lung adenocarcinoma A549 cells (1×10⁷)were intraperitoneally administered 4 times with intervals of 2 weeks.On the third day after the last immunization, the spleen was taken out.Spleen cells (1×10⁸) and murine myeloma X63-Ag8.653 cells (1×10⁷) weresubjected to cell fusion by the use of polyethyleneglycol (molecularweight, 1500) as the fusion promoting agent. The above cells werecultivated in a HAT culture medium (RPMI 1640 culture medium containinghypoxanthine, aminopterin, thymidine and 10% fetal calf serum) in aculture plate of 96 holes. After 1 week, the HAT culture medium wasexchanged with a fresh one (50 μl) once every 3 days. After 2 to 3weeks, 280 clones were observed in 400 wells. Screening was carried outusing the supernatants from these 280 wells.

Screening was effected by application of the indirect immuno-fluorescentantibody method and the indirect immuno-peroxidase method. The formermethod was performed by the use of the hybridoma supernatant as theprimary antibody and the FITC labeled rabbit anti-mouse immuno-globulinas the secondary antibody, and the reactivity with human lungadenocarcinoma A549 was investigated by the aid of a fluorescentmicroscope. Among the hybridoma cells associated with human lungadenocarcinoma A549 cells as used in immunization, the hybridoma YH206cells strongly reactive to lung adenocarcinoma on a tissue slice werechosen and subjected to cloning twice.

The latter method was performed by fixing the tissue materials obtainedfrom various cancers and fetal tissues with 10% formalin, embedding thefixed specimens in paraffin, selecting lung adenocarcinoma, lungalveolar cell carcinoma, lung epidermoid carcinoma and lung large cellcarcinoma and normal lung from the embedded specimens and examiningtheir reactivity according to the Watanabe et al. method ("EnzymeAntibody Method, Theory-Operation, Explanation and its Application",pages 33-39 (1984)).

EXAMPLE 2

Preparation of the monoclonal antibody YH206:

Cloning hybridoma YH206 was cultivated in a cell culture flask, and thesupernatant was collected. On the other hand, the cells of saidhybridoma (1×10⁷) were intraperitoneally administered to BALB/c micepreviously administered with pristane(2,6,10,14-tetramethylpentadecane), and after about 2 weeks, the asciticfluid was collected. Said supernatant and said ascitic fluid were saltedout with ammonium sulfate and purified by gel filtration with SephacrylS-300.

EXAMPLE 3

Determination of the immunoglobulin class of the monoclonal antibodyYH206:

The immunoglobulin sub-class of the monoclonal antibody YH206 wasdetermined to be IgM according to the Ouchterlony method by the use ofrabbit anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA and IgM (Miles Lab.).

EXAMPLE 4

Examination of the tissue distribution of the antigen corresponding tothe monoclonal antibody YH206:

Using the monoclonal antibody YH206 as obtained in Example 2, the tissuedistribution of the corresponding antigen was examined on various humancancer tissues, normal human adult tissues and fetal tissues.

Each tissue was fixed with 10% formalin, embedded in paraffin and slicedto make a specimen of 4 to 5 μm in thickness, followed by storage at 4°C. The specimen was subjected to hematoxylin-eosin staining and thenhistologically examined. Thereafter, the indirect immuno-peroxidasemethod was applied thereto according to said Watanabe et al. method.Namely, the endogenous peroxidase activity of the specimen was removedby treatment with 0.6% hydrogen peroxide-methanol. Normal rabbit serumwas reacted thereto for inhibiting the non-specific adsorption of thesecondary antibody. The monoclonal antibody as the primary antibody andthen the peroxidase-labled rabbit anti-mouse immunoglobulin were reactedthereto. Coloring was effected by the use of 3,3'-diaminobenzidine.Nuclear staining was carried out with 0.2% methylgreen.

The results are shown in Tables 1 and 2.

                  TABLE 1                                                         ______________________________________                                        Tissue distribution of the antigenic                                          determinant recognized by the                                                 monoclonal antibody YH206                                                     (A) Cancer tissues                                                                                      Staining                                            Organ    Type             property                                            ______________________________________                                        Lung     adenocarcinoma   ++(*)   (9/13)(**)                                           alveolar cell carcinoma                                                                        ++      (2/2)                                                epidermoid carcinoma                                                                           F+      (1/6)                                                large cell carcinoma                                                                           F+      (1/5)                                                small cell carcinoma                                                                           -       (0/6)                                       Pancreas adenocarcinoma   ++      (3/4)                                       Stomach  adenocarcinoma   +       (6/9)                                                singnet-ring cell carcinoma                                                                    -       (0/3)                                       Liver    cholangiocarcinoma                                                                             +       (1/1)                                                hepatocellular carcinoma                                                                       -       (0/2)                                       Breast   scirrhous carcinoma                                                                            -       (0/2)                                       Colon    adenocarcinoma   -       (0/10)                                      Kidney   Grawitz's tumor  -       (0/1)                                       Gall bladder                                                                           adenocarcinoma   -       (0/1)                                       ______________________________________                                         (*)Staining intensity: ++, strongly positive; +, positive; F+, faintly        positive; -, negative.                                                        (**)Number of positive/number of tested.                                 

                  TABLE 2                                                         ______________________________________                                        Tissue distribution of the antigenic                                          determinant recognized by the                                                 monoclonal antibody YH206                                                     (B) Normal tissues                                                            Organ        Adult             Fetus                                          ______________________________________                                        Lung         -(*)    (0/8)(**) ++      (1/1)                                  Colon        -       (0/9)     +       (1/1)                                  Kidney       F+      (3/4)     ++      (1/1)                                  Liver        -       (0/4)     +       (0/1)                                  Pancreas     F+      (3/3)     F+      (1/1)                                  Small intestine                                                                            -       (0/4)     +       (1/1)                                  Stomach      -       (0/8)     n.t.(***)                                      Adrenal gland                                                                              -       (0/1)     n.t.                                           Brain        -       (0/1)     n.t.                                           Esophagus    -       (0/2)     n.t.                                           Heart        -       (0/1)     n.t.                                           Lymphnode    -       (0/2)     n.t.                                           Skeltal muscle                                                                             -       (0/1)     n.t.                                           Spinal cord  -       (0/1)     n.t.                                           Spleen       -       (0/2)     n.t.                                           Thyroid gland                                                                              -       (0/1)     n.t.                                           ______________________________________                                         (*)Staining intensity: ++, strongly positive; +, positive; F+, faintly        positive; -, negative.                                                        (**)Number of positive/number of tested.                                      (***)Not tested.                                                         

In the case of lung cancers, a relatively limited reactivity toadenocarcinoma and alveolar cell carcinoma was indicated, and 11 caseswere positive in 15 cancers. The staining pattern of the adenocarcinomawas a labeling of the apical surface of tumor cell, while that of thelung epidermoid carcinoma was cytoplasmic. In the case of large cellcarcinoma, staining property was recognized in one of 5 cases, but itsobservation was limited to a part of cancer cells. In the case of smallcell carcinoma, no positive reaction was observed. In the case of otherorgan carcinomas, positive reaction was observed in 3 cases of 4pancreas cancers, 6 cases of 9 stomach cancers and 1 case of 1 gallbladder cancer. In stomach adenocarcinoma, most cases were positive inan apical surface and also in secreted products with a tendency thatmucin lake is strongly stained. On the other hand, stomach signet-ringcell carcinoma (0/3), liver hepatocellular carcinoma (0/2), largeintestine carcinoma (0/10), kidney cancer (0/1), breast cancer (0/2) andgall bladder adenocarcinoma (0/1) gave no reactivity.

As shown in Table 2, the examination of the non-malignant tissues ofhuman adults revealed that extremely limited reaction is slightlyrecognized with renal tubule and extrapancreatic secreting gland. In thelung tissue, no reaction was observed with any of alveolar epithelium,bronchial epithelium and tracheal mucous gland. Distribution of theantigen in the fetal tissue was so broad as including lung, and thestaining property was strong in comparison with that in the adulttissue.

As understood from the above, the antigen distribution is limited toadenocorcinoma and alveolar carcinoma in the lung tissue, while itextends to stomach cancer and pancreas cancer with high frequency amongother tissues. In the non-malignant adult tissue, weak reaction wasobserved only with very small portions of renal tubule epithelium andextrapancreatic secreting gland, and detection was not made from anynormal lung tissue. The antigen distribution in the fetal tissue extendsto a relatively broad area, and reactivity is observed in stomach, largeintestine and lung where the antigen is not detected in case of theadult tissue. The antigen is thus detected mainly in adrenocarcinoma andthe fetal tissues and only in trace from the normal adult tissues. Fromsuch characteristic distribution, it may be said that the antigencorresponding to the monoclonal antibody YH206 is like carcinoembryonicantigen (CEA).

EXAMPLE 5

Reactivity of the monoclonal antibody YH206 with erythrocytes:

A 2% suspension of human erythrocytes A₁, A₂, B or O (Ortho Diagnostics)was prepared, and the reactivity with the monoclonal antibody YH206 wasexamined with the hemagglutination according to the physiological salinesolution method, the albumin method and the indirect Coombs' test. Asthe positive control, the anti-H (type 2) monoclonal antibody S1 wasused. Further, the reactivity with neuraminidase-treated erythrocyteswas examined by the Rahman-Longenecker method.

Namely, neuraminidase (Sigma No. N-2876) was added to a 5% erythrocytesuspension in an amount of 0.5 μ/ml, and incubation was effected at 37°C. for 1 hour. After washing with PBS (phosphate buffered saline), itwas used in the form of a 2% suspension. As the positive control, theThomsen-Friedenreich antigen-monoclonal antibody 49H.8 was used. Theantibody was used by adjusting the purified product to a concentrationof 5 μg/ml.

As the result, the monoclonal antibody YH206 did not produce anyhemagglutination with any of the erythrocytes, and no correlation to anyof the A, B and H-type antigens was observed. Also, no agglutinationwith the neuraminidase-treated erythrocytes was produced.

EXAMPLE 6

Chemical properties of the antigen corresponding to the monoclonalantibody YH206 - periodic acid oxidation of tissue strips:

According to the known method as described in Kiernan: "Histological andHisto-chemical Methods--Theory and Practice", pages 153-156 (1981), eachof the tissue strips from various organs after removal of theendogeneous peroxidase activity was reacted with 1% periodic acidsolution at room temperature for 10 to 20 minutes, followed by stainingas in Example 4.

As the result, it was observed that the tissue having a reactivity withthe monoclonal antibody YH206 loses the reactivity with thecorresponding antigen completely when oxidized with periodic acid. Thisfact suggests that the antigenic determinant of the antibody is a sugarchain.

EXAMPLE 7

Chemical properties of the antigen corresponding to the monoclonalantibody YH206 - neuraminidase digestion of tissue strips:

In order to investigate whether sialic acid participates in theantigenic determinant, observation was made on the reactivity of thetissue strips with the monoclonal antibody YH206, said tissue stripsconsisting of 13 malignant tissues and 8 non-malignant tissues givingpositive or negative in the immuno-peroxidase method previously digestedwith neuraminidase. Neuraminidase digestion was carried out according tothe Atkinson et al method. Namely, the tissue strips after removal ofthe endogenous peroxidase activity were treated with neuraminidase(Sigma No. N-3001) (1 μ/ml; 0.1M acetate buffer of pH 5.11) at 37° C.for 12 hours, washed well with PBS and subjected to immuno-peroxidasestaining. For the tissue strips as the control, 0.1M acetate buffer notcontaining neuraminidase was used.

As the result, the decrease of the intensity or frequency of thestaining positivity after neuraminidase digestion was not observed.Rather, the increase of staining with neuraminidase treatment wasobserved in some cases. In 12 cases of lung cancer, stomach cancer andlarge intestine cancer, the antibody not detected before treatment wassometimes detected after neuraminidase digestion. The lung alveolar cellcarcinoma which was negative before treatment showed positive afterneuraminidase digestion. The lung large cell carcinoma from which thecorresponding antigen was not detected by the immuno-peroxidase methodshowed the remarkable presence of the antigen-positive cells afterneuraminidase digestion, and this phenomenon was also observed in lungepidermoid carcinoma. The similar tendency to the above was alsoobserved in 3 cases among 8 non-malignant tissues. In the gastric mucosaafter neuraminidase digestion, the antibody showed reactivity to thegastric glands as well as the gastric epithelium. From these facts, itmay be said that in at least some of the malignant and non-malignanttissues, the antigen is masked with sialic acid and becomes non-maskedon treatment with neuraminidase.

This can be confirmed not only by the test with the tissue strips butalso by the test with non-fixed cultured cancer cells. Accordingly, theantigenic determinant may be considered to be present in a cryptic format the non-cancerous parts and masked with sialic acid. At the cancerousparts, the antigenic determinant is disclosed as such so that themonoclonal antibody may be detected with high frequency.

EXAMPLE 8

Chemical properties of the antigen corresponding to the monoclonalantibody YH206 - inhibition test of tissue strips with lectins:

For investigation of the antigen determinant, inhibition test waseffected by the use of various lectins.

According to the known method as described in Hirano: "HistologicalChemistry 1981--histological application of lectins", pages 17-39(1982), each of biotinated lectins such as Ulex europeus, Canavaliaensiformis, Ricinus communis, Triticum vulgaris, Dolichos biflorus andArachis hypogaea (Vector Labs.) was adjusted to a concentration of 50μg/ml and subjected to reaction with tissue strips for 30 minutes. Thereactivity of the monoclonal antibody was investigated as in Example 4.

As the result, any of the lectins as used could not completely block thereaction of the monoclonal antibody YH206. But, wheat-germ aggulutininshowed slight decrease of the reactivity of YH206.

The above fact suggests that a sugar portion (N-acetylglucosamine orsialic acid) reactive to WGA may be present in the antigenicdeterminant.

EXAMPLE 9

Chemical properties of the antigen corresponding to the monoclonalantibody YH206 - enzymatic treatment of cultured cells:

It was investigated whether the decrease or disappearance of the antigendeterminant is caused by treatment with a protease such as tripsin orprotease V 8. Namely, cultured cells (2×10⁶) of lung adenocarcinoma A549were suspended in an RPMI 1640 culture medium (1 ml) and reacted withany enzyme solution (1 ml) as hereinafter mentioned at 37° C. for 1hour. Then, a 10% FCS added RPMI 1640 culture medium (8 ml) was addedthereto to stop the reaction, washed well with RPMI and subjected toobservation on the reactivity with the monoclonal antibody according tothe indirect immunofluorescent antibody method. As said enzyme solution,there were used the ones chosen from the following five kinds:neuraminidase F (Seikagaku Kogyo) (0.1 μ/ml), protease V 8 (Miles) (0.5mg/ml), trypsin (Warsinton) (0.5 mg/ml), glycosidase (mixed) (SeikagakuKogyo) (2 mg/ml) and endoglycosidase H (Seikagaku Kogyo) (0.1 μ/ml). Nodecrease or disappearance of the antigen determinant was observed bytreatment with any of the above five kinds of enzymes. This factsupports that the antigen determinant may be a sugar chain.

EXAMPLE 10

Detection and immunochemical investigation of the corresponding antigenin the lung adenocarcinoma supernatant:

Extraction of the crude antigen:

A549 cell (1×10⁷) were cultivated in a low concentration (1%) FCS addedculture medium (HAM culture medium (Flow Laboratories), 50%; RPMI 1640(Difco), 50%; hepes (Sigma), 20 mM; penicillin, 100 mg/liter; kanamycin,50 mg/liter; sodium bicarbonate, 1 g/liter; insulin (Sigma), 5 mg/liter;transferrin (Sigma), 35 mg/liter; phosphatidylethanolamine (Sigma), 0.5mg/liter; sodium selenite, 4.3×10³ mg/liter) (70 ml) for 3 days. Thesupernant was collected several times and used as a crude antigen. Fromcultured lung cancer A549 cells and large intestine cancer BM314 cells,the crude antigen was extracted with 1% NP 40 (Nakarai Chemical)according to the freezing and thawing method. For fetal faeces, 1% NP 40was used.

Enzyme antibody method:

The culture supernatant prepared according to the Magnani et al. method(J. Biol. Chem., 257, 14365-14369 (1982)) was concentrated to make a 2.5fold, 10 fold, 20 fold or 40 fold concentration, which was admitted intoa round bottom microplate (Falcon) in an amount of 50 μl/well andreacted at 37° C. for 12 hours. After blocking with 3% bovine serumalbumin, the monoclonal antibody was reacted thereto. Then,peroxidase-conjugated rabbit anti-mouse immunoglobulin was reactedthereto, coloring was made with o-phenylenediamine, and absorbance wasmeasured at 492 nm.

SDS-PAGE and western blotting:

The operation was carried out according to the Imai et al. method(Cancer Research, 41, 1028-1033 (1981)). As the gel, 5% or 10% gel wasused. The antigen (50 μl) was developed with SDS-PAGE, transferred fromthe gel to the nitrocellulose membrane (pore size, 0.45 μ) according tothe Watanabe et al. method ("Immunological Experimental Operations", 11,3485-3489 (1982)) and detected with the indirect enzyme antibody method.

Gel-filtration:

As the gel, there was used Sephacryl S-300 (2.4 cm×80 cm). On eachfraction obtained by the use of the antigen (3 ml) as the startingmaterial, the reactivity with the monoclonal antibody was measuredaccording to the enzyme antibody method. For detection of the antigen inthe supernatant of A549 cells, the enzyme antibody method using theantibody YH206 was applied. As the result, it was revealed that, in thesupernatant of A549 cells, the antigen molecule reactive to YH206 ispresent. The supernatant was subjected to gel filtration using SephacrylS-300, and the reactivity of the antigen of each fraction was measuredby the enzyme antibody method. As the result, the antigen was detectedaround the void volume.

Then, the molecular weight of the corresponding antigen was investigatedusing A549 cells, CEA-producing large intestine cancer BM314 cells andfetal faeces as the crude antigen. As the control of the antibody, theanti-CEA monoclonal antibody AS802 was used. AS802 showed the reactionwith fetal faeces and CEA-producing large intestine cancer BM314 cellsat the position of about 200K dalton. On the other hand, YH206 showedthe reaction with fetal faeces in three bands at the position of ahigher molecule than CEA but did not show any significant reaction withthe antigen obtained by extraction of the immunogen A549 with NP40.

Then, the analysis was made on the antigen obtained by extraction fromA549 cells by the freezing and thawing method and the A549 supernatantas the crude antigen in the same manner as above. When the extractionwas effected by the freezing and thawing method or when the A549supernatant was used as the crude antigen, a clear single band wasobserved at the position of such a high molecule as more than 330Kdalton.

Thus, the presence of an antigen in the A549 supernatant was shown bythe enzyme antibody method, and SDS-PAGE and western blotting wereeffected with the antigen so that its molecular weight was revealed tobe more than 330K dalton. Further, the reactivity of the antigen of eachfraction obtained by gel filtration with Sephacryl S-300 was measured bythe enzyme antibody method, and the antigen was detected around the voidvolume. Also, fetal faeces were analyzed by SDS-PAGE and westernblotting, and three bands were observed at the position of a highermolecule than CEA; these are different from the antigen in thesupernatant with respect to the molecular weight.

EXAMPLE 11

Detection of the antigen in a blood stream:

Since the antigen is present in the supernatant of the culture medium,it is expected that the antigen may be also present in a blood stream.The serum of a cancer patient was thus examined according to the RPHA(reversed passive hemagglutination) method.

The RPHA method was performed as reported by Iguro et al. (Lancet, 6,817-818 (1984)). Namely, inactivated serum was absorbed with sheep redblood cells (SRBC) (room temperature, 1 hour) and serially diluted withPBS. The resulting dilutions were each added to a round bottommacro-titer plate (Sanko Pure-Chemical) in an amount of 30 μl per well.An equal quantity of a 2% suspension of SRBC associated with themonoclonal antibody with chromium chloride was added thereto, allowed tostand at room temperature for 4 hours and then subjected to observationon the hemagglutination. By previous addition of the monoclonal antibodyto the serum or the antigen solution prepared as in Example 10, it wasconfirmed that the hemagglutination of SRBC is inhibited with adependency on the antigen concentration.

As the test sera, there were used a total of 178 cases comprising 30cases of normal control, 67 cases of lung cancer, 20 cases of stomachcancer, 15 cases of pancreas cancer, 15 cases of large intestine cancerand 31 cases of benign diseases (pneumonia, 5 cases; chronic hepatitis,6 cases; liver cirrhosis, 7 cases; chronic articular rheumatism, 2cases; peptic ulcer, 11 cases).

For the fundamental examination, a concentrate of the supernatant of theculture of A549 was used as the antigen, and hemagglutination wasobserved even when diluted to 4096 folds. Then, the above antigen wasserially diluted and reacted with the antibody YH206, followed byhemagglutination. As the result, it was confirmed that thehemagglutination value was confirmed to be inhibited depending on theamount of the antibody as added. Similar results were obtained when thepatient serum was used as the antigen.

On the basis of the above fundamental examination, the antigen in theblood taken from each of the patients bearing cancers was measured bythe RPHA method. The level of the dilution of the serum for antigenpositive was tentatively decided to be 1/64 dilution or more. In 30healthy donors, three sera (10%) had antigen levels of 1/64 dilution. Incontrast, among 67 patients with lung cancer, 35 sera (52.2%) showedpositive. Further, 9 sera (45%) in 20 patients with stomach cancer, 11sera (73.3%) in 15 patients with pancreas cancer and 9 sera (60%) in 15patients with colon cancer indicated positive. When the sera frompatients bearing no cancer were used, 4 sera (12.9%) in 31 patients,i.e. 2 sera in 7 patients with liver cirrhosis and 2 sera in 5 patientswith acute pneumoniae (CRP being not less than 5+), were positive; allof 6 patients with chronic liver hepatitis, 2 patients with chronicarticular rheumatism and 11 patients with peptic ulcer were negative;and in positive cases, hemagglutination was observed so low as 64 fold(1/64 dilution).

Observation on the sera from patients with lung cancer, of which thehistological type was previously clarified, gave positive results for 13cases (61.9%) in 21 adenocarcinoma, 6 cases (40%) in 15 epidermoidcarcinoma, 8 cases (50%) in 16 small cell carcinoma and 3 cases (75%) in4 large cell carcinoma.

Since SRBC was used as the indicater cells, the Forssman antibodytherein might be detected. Therefore, the absorption with SRBC waspreviously applied. In case of sera having a high hemagglutinationactivity, it was confirmed that no hemagglutination would not take placewith SRBC associated with YH206.

Summarizing the above results, it may be noted that while weak positivereaction is observed only in 3 cases among 30 healty donors, thepercentage of positivity in various cancer patients is so high as 45 to75% and the titers are relatively high in most cases.

From the above results, it is thus clear that the antibody of theinvention is useful as a reagent for immunoassay of the correspondingantigen in a body fluid, particularly for diagnosis of various cancers.

EXAMPLE 12

Relationship to the clinical symptomatic stage of lung cancer:

With the lung cancer of which the clinical symptomatic stage ispreviously clarified, the amount of the antigen in blood was examined.The positivity percentage of the progressive cancers at the stages IIIand IV (73.3% and 62.5%) was higher than that at the stage I (22.2%).Further, the increase of the antigen amount in the former was higherthan that in the latter.

Human lung adenocarcinoma cell line A549 and hybridoma YH206 have beendeposited under the terms of the Budapest Treaty with the FermentationResearch Institute, Agency of Industrial Science and Technology,Ibaragi-ken, Japan and have respectively assigned Accession Numbers FERMBP-2000 and FERM BP-2001.

The deposit of A549 and the deposit of YH206 were both made on Aug. 16,1988.

What is claimed is:
 1. A cell line having all the identifyingcharacteristics of YH206.
 2. A monoclonal antibody produced by the YH206cell line.